ORIGINAL PAPER In vitro antifungal and antibacterial ...

Chemical Papers 63 (5) 543–547 (2009) DOI: 10.2478/s11696-009-0049-z

ORIGINAL PAPER

In vitro antifungal and antibacterial properties of thiodiamine transition metal complexes a,b

Ajay K. Mishra*, a Shivani B. Mishra, b Narender K. Kaushik

a Department

of Chemical Technology, University of Johannesburg, Doornfontein-2028, South Africa b Department

of Chemistry, University of Delhi, Delhi-110007, India

Received 9 October 2008; Revised 1 December 2008; Accepted 4 December 2008

Synthesis, characterization and biological studies of some thiodiamine metal complexes are described. Cobalt(II) and copper(II) complexes of type [Cu(L)2 Cl2 ] and [Co(L)2 SO4 ], where L = (cyclohexyl-N-thio)-1,2-ethylenediamine (L1 ) and (cyclohexyl-N-thio)-1,3-propanediamine (L2 ), were synthesized. The synthesized copper and cobalt thiodiamine complexes were characterized by elemental analysis, IR, mass, UV-VIS and 1 H NMR spectroscopic studies. Thiodiamines coordinate as a bidentate N—S ligand. The binding sites are azomethine nitrogen and thioamide sulfur. Molar conductance values in dimethylsulfoxide indicate non-electrolyte nature of the complexes. In vitroantimicrobial screening shows promising results against both bacterial and fungal strains. c 2009 Institute of Chemistry, Slovak Academy of Sciences Keywords: synthesis, thiodiamine, spectral, antibacterial, antifungal

Introduction Much attention has been paid to biologically active metal complexes in recent years. Sulfur and nitrogen donor ligands have been widely studied due to their high potential to coordinate with transition metals. Compounds containing thione and thiol groups have important position among organic reagents as potential donor ligands for the transition metal ions (Ali & Livingstone, 1974). Organic compounds and metal complexes both display a wide range of pharmacological activity including anticancer, antibacterial, and fungistatic effects. Several papers on coordination chemistry of thiohydrazide, thiodiamine and thiohydrazone have been published (Mishra et al., 2006a, 2007). Ligands with sulfur and nitrogen donor atoms in their structures act as good chelating agents for the transition and non-transition metal ions (Biswas et al., 1984; Kaushik & Mishra, 2003). Coordination of such compounds with metal ions, e.g. copper, nickel, and iron, often enhance their activi-

ties, as reported for pathogenic fungi (Singh et al., 2000). Thiodiamines in the present investigation were used with cyclohexylamine. The functional group provided a “point of attachment”, a system which mimics certain classes of biological systems and in addition, such groups could be toxic to microbes when used as drugs. Interest in complexes of these ligand systems covers several areas ranging from general considerations of the effect of sulfur and electron delocalization in transition metal complexes to potential biological activity and practical applications (Padhye & Kauffman, 1985). The aim of the present work was to synthesize and characterize complexes of cobalt(II) and copper(II) with potential active thiodiamine ligands. Both ligands and complexes were characterized by elemental analysis, IR, mass, electronic, and 1 H NMR spectroscopic studies. Molar conductivity was determined for these complexes. In vitro antibacterial and in vitro fungal studies were also carried out for these synthesized complexes.

*Corresponding author, e-mail: [email protected]

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A. K. Mishra et al./Chemical Papers 63 (5) 543–547 (2009)

Fig. 1. Synthesis of cyclohexyl-N-thiodiamines and thiodiamine [Co(L)2 SO4 ] and [Cu(L)2 Cl2 ] complexes.

Experimental All reagents used were of the AR grade. Elemental analysis of ligands and metal complexes were done on an Elementar Analysensysteme Gmbh Vario El-III. IR was recorded on a Perkin-Elmer spectrum 2000 FTIR spectrometer using KBr discs. Electronic spectra were recorded on a Shimadzu UV-VIS spectrophotometer Model 1601 using acetone as the solvent. Conductance measurements were carried out on a Digital Conductometer Model PT-827, India, using acetone as the solvent. A Model Jeol SX102/DA-600 (KV 10MA) was used to record mass spectra of the ligands in acetone solvent. 1 HNMR was recorded on a Brucker spectrospin 300 spectrometer using deuterated acetone. Cyclohexyl-N-thiodiamines were synthesized using methods described previously (Mishra & Kaushik, 2007). Fig. 1 summarizes the synthesis conditions. Preparation of thiodiamine [Co(L)2 SO4 ] and [Cu(L)2 Cl2 ] complexes (L = L1 or L2 ) started by dissolving the corresponding ligand L (0.101 g, 0.5 mmol of L1 , or 0.108 g, 0.5 mmol of L2 ) in methanol and addition of a few drops of 1 mol L−1 HCl solutions of the corresponding metal salt (0.070 g, 0.25 mmol of cobalt sulfate, or 0.043 g, 0.25 mmol of cupric chloride). The solution was stirred for 4–5 h. Greenishblue color of the copper complex and green color of the cobalt complex appeared immediately, the complexes were separated, washed with ether and dried in a desiccator over CaCl2 under vacuum. Cobalt and copper complexes synthesis is schematically shown in Fig. 1. Most compounds were screened in vitro against Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. Among several methods available, the common method of Rajesh and Sharma (2002), was adopted. Susceptibility of fungi to various fractions of compounds was assayed by the microbroth dilution method. The Sabouraud dextrose medium was dissolved in glass double distilled water and autoclaved at 70 kPa for 15 min. A volume of 90 µL of the medium was added to the wells of the cell culture

plates (Nunc Nunclon). Different concentrations in the range of 1000-10 µg mL−1 of various fractions were prepared in duplicate wells and then, the wells were incubated with 10 µL of conidial suspension containing 1 × 104 conidia. The plates were incubated at 37 ◦C and examined macroscopically after 48 h for the growth of Aspergillus mycelia. The activity was represented by concentration (µg mL−1 ) against all fungal strains: Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. The basic method for spore germination inhibition was modified and used to evaluate the activity of various test fractions against fungi. Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were grown on Sabouraud dextrose agar plates and their homogenous conidial suspension was prepared in the Sabouraud maltose broth. The conidia were counted and their number in the suspension was adjusted to 1 × 104 mL−1 . Various concentrations in the range of 1000–10 µg mL−1 of the test samples in 90 µL of the culture medium were prepared in 96-well flat bottom micro-culture plates (Nunc Nunclon) by the double dilution method. The wells were prepared in duplicates for each concentration. The wells were inoculated with 10 µL of conidial suspension containing (100 ± 5) conidia. The plates were incubated at 37 ◦C for 10 h and then examined for spore germination under an inverted microscope (Nikon Diphot). The number of germinated and non-germinated conidia was recorded. Most compounds were screened in vitro against the bacterial strains Staphylococcus epidermidis (S. epidermidis), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). Various methods are available for the evaluation of antibacterial activity of different types of drugs. However, the most widely used method (Eldeen et al., 2005) consists in the determination of the antibacterial activity of the drug. The disc diffusion assay was used to determine antibacterial activity of the drug using gram positive and gram negative strains of bacteria, namely of S. epi-


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Table 1. Elemental analysis of thiodiamine complexes wi found (calculated)/% Complex C Co(L1 )2 SO4 Cu(L1 )2 Cl2 Co(L2 )2 SO4 Cu(L2 )2 Cl2

38.77 40.21 40.92 42.50

(38.71) (40.26) (40.96) (42.52)

H 6.80 7.06 7.16 7.42

N

(6.81) (7.08) (7.17) (7.44)

15.10 15.62 14.35 14.91

S

(15.05) (15.66) (14.33) (14.88)

17.25 11.98 16.35 11.30

(17.20) (11.93) (16.38) (11.34)

Cl – 13.16 (13.23) – 12.55 (12.58)

Ma 10.82 11.80 10.28 11.22

(10.75) (11.84) (10.24) (11.25)

a) Metal.

dermidis, S. aureus, E. coli, and P. aeruginosa. Base plates were prepared by pouring 10 mL of autoclaved Muller–Hinton agar (Biolab) into sterile Petri dishes (9 cm) and allowing them to settle. Molten autoclaved Muller–Hinton that had been kept at 48 ◦C was inoculated with a broth culture (106 –108 mL−1 ) of the test organism and then poured over the base plate. The discs were air dried and placed on the top of the agar layer. Four replicates of each drug were tested (four discs per plate) using a gentamycin disc (0.5 µg per disc) as a reference. The plates were then incubated for 18 h at room temperature. Antibacterial activity is expressed in the form of the zone of inhibition (mm). The serial dilution technique, using 96-well micro plates to determine the minimum inhibitory concentration (MIC) of drugs for antibacterial activity was used. The 2 mL cultures of four bacterial strains: S. epidermidis, S. aureus, E. coli, and P. aeruginosa, were prepared and placed in a water bath for overnight at 37 ◦C. The overnight cultures were diluted with sterile Muller–Hinton broth. The drugs were resuspended to a concentration of 60 µg per disc (in DMSO) with sterile distilled water in a 96-well micro plate. A similar two fold serial dilution of the reference drug gentamycin (Sigma) was used as a control against each bacterial strain. 100 µL of each bacterial culture (106 – 108 mL−1 ) were added to each well. The plates were covered and incubated overnight at 37 ◦C. To indicate bacterial growth, p-iodonitrotetrazolium violet was added to each well and the plates were incubated at 37 ◦C for 30 min. Bacterial growth in the wells was indicated by red color, whereas clear wells indicated inhibition.

Result and discussion Characterization of ligands and complexes Elemental analysis (Table 1) revealed the purity of the complexes. All complexes are soluble in DMF and DMSO. Molar conductance values of the isolated complexes measured in DMSO were found to be less than 15 Ω−1 cm2 mol−1 suggesting their non-electrolytic nature. Distinguishable absorption bands of electronic spectra of the complexes in a DMSO solution were

Table 2. Electronic spectra of the ligands and complexes λmax /nm

log ε

L1

212 254 285

3.95 3.22 2.70

Co(L1 )2 SO4

295 381 485 664

4.27 3.78 3.34 2.78

Cu(L2 )2 Cl2

282 378 480 644

4.16 3.93 3.54 2.98

L2

213 242 287

4.33 3.96 2.56

Co(L2 )2 SO4

300 376 482 656

4.28 3.62 2.80 2.37

Cu(L1 )2 Cl2

286 356 475 667

4.41 3.53 2.99 2.91

Compound

recorded and are listed in Table 2. Both thiohydrazides and thiodiamines ligands and their metal complexes show high intensity absorption in the UV region. These absorptions are due to the chromophore N—C—S group. An absorption band at about 280 nm was observed and assigned to a π–π ∗ electronic transition. The electronic spectra of cobalt(II) and copper(II) complexes show d–d bands between 610–660 nm. In addition to the intra-ligand and d–d bands, the complex also exhibits a strong band between 440– 480 nm which can safely be assigned to the ligand to metal charge-transfer transition (Ali et al., 2001). IR spectra of the ligands and metal complexes are summarized in Table 3. Strong bands at 2900– 3250 cm−1 in the thiohydrazide and thiodiamine metal


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Table 3. IR spectra of the ligands and complexes ν˜ /cm−1 Compound (N—H) L1 Co(L1 )2 SO4 Cu(L1 )2 Cl2 L2 Co(L2 )2 SO4 Cu(L2 )2 Cl2

2924 3186 2931 3185 2936 3143 2924 3249 2920 3163 2931 3156

Thioamide I

Thioamide II

(N—N)

(C— —S)

(M—N)

(M—S)

(M—Cl)

(M—O)

1464

1310

1025

886

–

–

–

–

1500

1339

1022

845

467

373

–

415

1506

1336

1027

868

460

370

295

–

1463

1330

1028

889

–

–

–

–

1505

1335

1022

845

485

375

–

418

1516

1327

1032

810

480

381

295

–

complexes were assigned to the —NH stretching vibrations of NH2 and NH groups. The NH stretching of ligand was found to be shifted to higher frequencies, the change being associated with coordination of terminal NH2 nitrogen to the metal ion. Bands located at 1460 cm−1 , 1307 cm−1 , 1023 cm−1 , and 875 cm−1 were assigned to the thioamide-I, -II, -III, and -IV vibration, respectively. A sharp and strong band between 860-890 cm−1 in the IR spectra of free ligands, which is shifted in the complexes, was attributed to (C—S) (Mishra et al., 2006a; Mishra & Kaushik, 2007; Choudhary et al., 1998). In the spectra of cobalt(II) and copper(II) complexes, the metal nitrogen vibrations (M—N) were assigned to the new bands in the far IR between 470–490 cm−1 , while in the region between 370–390 cm−1 , metal–sulfur gave a (M—S) band stretching (Aggarwal & Rao, 1978). The band at 280–300 cm−1 was assigned to the (Cu— Cl) stretching vibrations. The band at 410–420 cm−1 was assigned to the (Co—O), SO4 bidentate stretching vibration. 1 H NMR spectra of the ligands were recorded in d6 DMSO taking TMS as internal standards. L1 : δ: 8.9 (br-s, 1H, NH), 3.8 (br s, 2H, NH2 ), 2.42 (t, 2H, CH2 ), 1.16–1.8 (m, 11H, cyclohexyl). L2 : δ: 9.09 (br s, 1H, NH), 4.3 (br s, 2H, NH2 ), 2.2 (t, 4H, CH2 ), 1.4 (m, 2H, CH2 ), 1.16–1.77 (m, 11H, cyclohexyl). 1 H NMR spectra of the thiodiamine ligands (Mishra et al., 2006b, 2007; West et al., 1999) show signals at δ 9.00 and δ 3.26 due to the presence of NH protons. Two signals at δ 2.90 and δ 1.50 show the presence of a —CH2 group in the ligands. A peak between δ 7.2–6.6 for the case of ligand L1 clearly showing the presence of aromatic ring was also observed. In vitro antibacterial study In vitro antibacterial studies of the complexes are presented in Table 4. These synthesized complexes

Table 4. In vitro antibacterial studies of thiodiamine complexes, (–) denotes no activity Zone of inhibition/mm Complex S. epidermidis S. aureus E. coli P. aeruginosa Co(L1 )2 SO4 Cu(L1 )2 Cl2 Co(L2 )2 SO4 Cu(L2 )2 Cl2 Gentamycin

– 8 – –

– 8 – –

9 10 8 8

8 10 8 8

16

were tested against pathogenic bacterial strains such as S. epidermidis, S. aureus, E. coli, and P. aeruginosa using the disc diffusion method. Gentamycin was used as the reference drug for bacteria. In general, the compounds showed significant antibacterial activity against the bacterial strains with the zone of inhibition between 8–10 mm. In vitro antifungal study In vitro antifungal studies are summarized in Table 5. The complexes synthesized were tested against pathogenic fungal strains such as Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. Amphotericin B was used as the reference drug for fungi. The minimum inhibitory concentrations (MICs) by microbroth dilution assays (MDA) and the percent spore germination inhibition assays (PSGIA) are 31.25–500.00 µg mL−1 . Complexes [Cu(L2 )2 Cl2 ] and [Co(L2 )2 SO4 ] had the highest in vitro antifungal activity against all pathogenic fungal strains tested. Other complexes showed moderate activity probably due to the fact that Aspergillii have a hard chitinous outer wall and therefore higher concentration of fungicidal compounds may be often required to inhibit the fungi growth.


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Table 5. In vitro antifungal studies of thiodiamine complexes A. fumigatus Complex

Co(L1 )2 SO4 Cu(L1 )2 Cl Co(L2 )2 SO4 Cu(L2 )2 Cl2 Amphoterin B

A. flavus

A. niger

MDAa (µg mL−1 ) PSGIb (µg mL−1 ) MDA (µg mL−1 ) PSGI (µg mL−1 ) MDA (µg mL−1 ) PSGI (µg mL−1 ) 125 250 31.25 62.5 5

125 250 31.25 62.5 5

125 250 31.25 62.5 5

125 250 31.25 62.5 5

250 500 31.25 125 5

250 500 31.25 125 5

a) MDA = micro dilution activity; b) PSGI = percent spore germination inhibition.

Conclusions Spectral studies indicate that complexation to the metal ion proceeds through azomethine nitrogen and thioamide sulfur. In vitro antifungal activity of the complexes as compared with the standard drug Amphotericin B shows significant activity. The minimum inhibitory concentrations (MICs) by microbroth dilution assays (MDA) and percent spore germination inhibition assays (PSGIA) were found to be 31.25– 500.00 µg mL−1 . In vitro antibacterial study of the complexes as compared with the standard drug Gentamycin shows good activity. Bacterial strains with the zone of inhibition were observed between 8–10 mm. It is worth to mention that trials to get crystal suitable for X-ray structure determination failed due to amorphous character of the complexes. On basis of spectroscopic studies, the structure of complexes was proposed (Fig. 1). References Aggarwal, R. C., & Rao, T. R. (1978). Synthesis and structural studies of some first row transition metal complexes of acetone isonicotinoyl hydrazone. Journal of Inorganic and Nuclear Chemistry, 40, 171–174. DOI: 10.1016/00221902(78)80334-0. Ali, M. A., & Livingstone, S. E. (1974). Metal complexes of sulphur-nitrogen chelating agents. Coordination Chemistry Reviews, 13, 101–132. DOI: 10.1016/S0010-8545(00)80253-2. Ali, M. A., Mirza, A. H., Butcher, R. J., Tarafder, M. T. H., & Ali, M. A. (2001). Synthetic, spectroscopic, biological and X-ray crystallographic structural studies on a novel pyridinenitrogen-bridged dimeric nickel(II) complex of a pentadentate N3 S2 ligand. Inorganica Chimica Acta, 320, 1–6. DOI: 10.1016/S0020-1693(01)00452-2. Biswas, G. D, Biswas, P. K., & Chaudhuri, N. R. (1984). Conformational changes of nickel(II) diamine complexes in the solid state. Journal of Chemical Society, Dalton Transactions, 1984, 2591–2598. DOI: 10.1039/DT9840002591. Choudhary, R. K., Yadav, S. N., Tiwari, H. N., & Mishra, L. K. (1998). Structural aspects of morpholine-N-thiohydrazones complexes with some bivalent metals. Journal of Indian Chemical Society, 75, 392–394.

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